Thermo Fisher Scientific Pacific Blue Annexin V/SYTOX AADvanced Apoptosis Kit User guide

Paciﬁc Blue™ Annexin V/SYTOX™ AADvanced™ Apoptosis Kit for

Flow Cytometry

Catalog Number A35136

Pub.No. MAN0002443 Rev. A.0

WARNING! Read the Safety Data Sheets (SDSs) and follow the handling instructions. Wear appropriate protective eyewear,

clothing, and gloves. Safety Data Sheets (SDSs) are available from thermoﬁsher.com/support.

Product description

Annexins are a family of calcium-dependent phospholipid-binding proteins that preferentially bind phosphatidylserine (PS). Under normal

physiologic conditions, PS is predominantly located in the inner leaﬂet of the plasma membrane. Upon initiation of apoptosis, PS loses

its asymmetric distribution across the phospholipid bilayer and is translocated to the extracellular membrane leaﬂet marking cells as

targets of phagocytosis. Once on the outer surface of the membrane, PS can be detected by ﬂuorescently labeled Annexin V in a

calcium-dependent manner.

The Paciﬁc Blue™ Annexin V/SYTOX™ AADvanced™ Apoptosis Kit provides a rapid and convenient assay for apoptosis. The kit contains

recombinant annexin V conjugated to the violet-excitable ﬂuorophore, Paciﬁc Blue™ dye, to provide the maximum sensitivity. Because

Paciﬁc Blue™ dye absorbs maximally at 415nm with ﬂuorescence emission 455 nm, it is a good choice for violet diode laser excitation

in ﬂow cytometry. The kit includes the red ﬂuorescent dye, SYTOX™ AADvanced™ Dead Cell Stain, for identifying necrotic cells based on

membrane integrity. After staining a cell population with Paciﬁc Blue™ annexin V and SYTOX™ AADvanced™ stains in the supplied binding

buer, apoptotic cells show bright violet ﬂuorescence, dead cells show red ﬂuorescence, and live cells show dim violet ﬂuorescence

(Figure1). Because there is very little spectral overlap between the two dyes, very little or no compensation is required. We have

optimized this assay using Jurkat cells, a human T-cell leukemia line, treated with camptothecin to induce apoptosis. Some modiﬁcations

may be required for use with other cell types.

Figure1Jurkat cells (human T-cell leukemia) untreated control (panel A) or treated with 10 µM camptothecin for four hours (panel B).

Cells were treated with the reagents in the kit and analyzed by ﬂow cytometry using 405 nm and 488 nm excitation. Note that the camptothecin-

treated cells have a higher percentage of apoptotic cells (panel B) than the basal level of apoptosis seen in the control cells (panel A). A =

apoptotic cells, V = viable cells, N = necrotic cells.

Because no single parameter deﬁnes apoptosis in all systems, we strongly suggest using a combination of dierent measurements for

reliable detection of apoptosis. Refer to thermoﬁsher.com/apoptosis for a wide selection of products for apoptosis research.

USER GUIDE

For Research Use Only. Not for use in diagnostic procedures.

Contents and storage

Component Amount[1] Storage[2]

Paciﬁc Blue™ Annexin V (Component A)[3] 250 pL solution in 25mM HEPES, 140mM

NaCl, 1 mM EDTA, pH 7.4, with 0.1% bovine

serum albumin (BSA)

Store at 2–6°C.

Protect from light.

Do not freeze.

SYTOX™ AADvanced™ Dead Cell Stain

(Component B)[4] 1 vial Store at 2–6°C.

Protect from light.

Dimethylsulfoxide (DMSO), anhydrous

(Component C) 100µL

Store at 2–6°C.

5X Annexin-binding buer (Component D) 15mL

[1] Sufficient material is supplied for 50 assays, based on the protocol below.

[2] When stored as directed, this kit is stable for at least 6months.

[3] Approximate fluorescence excitation/emission maxima: 415/455 nm

[4] Approximate fluorescence excitation/emission maxima: 546/647 nm, bound to DNA

Spectral characteristics

The ﬂuorescence excitation and emission spectra of the Paciﬁc Blue™ annexin V conjugate (Component A) has ﬂuorescence

excitation/emission maxima of 415 nm and 455 nm, respectively.

The ﬂuorescence excitation and emission spectra of the SYTOX™ AADvanced™ Dead Cell Stain (Component B) were obtained from

samples of the dye bound to DNA. The SYTOX™ AADvanced™ Dead Cell Stain exhibits a ﬂuorescence enhancement of greater than

500‑fold. The SYTOX™ AADvanced™ Dead Cell Stain/DNA complex has ﬂuorescence excitation and emission maxima of 546nm and

647 nm, respectively.

Required materials not supplied

• Flow cytometry tubes

• Cells and culture medium

• Deionized water

Procedural guidelines

• We have optimized this assay using Jurkat cells treated with camptothecin to induce apoptosis. Some modiﬁcations may be required

for use with other cell types.

• Make sure that all tubes contain the same number of cells for a given experiment. Tube-to-tube variation in cell number leads to

signiﬁcant dierences in staining, and such variation can aect results.

• We recommend phosphate-buered saline (PBS) for suspending cells during staining.

• Do not use glass containers or tubes.

Label apoptotic cells for ﬂow cytometry

1. Remove the kit from the refrigerator, and allow the contents to equilibrate to room temperature.

2. Prepare 1X annexin-binding buer. For example, for ∼10 assays, add 1 mL of 5X annexin-binding buer (Component D) to 4mL of

deionized water.

3. Prepare 500µM SYTOX™ AADvanced™ Dead Cell Stain working solution by adding 200 µL DMSO (Component C) to one vial of

SYTOX™ AADvanced™ Dead Cell Stain (Component B). Mix the solution well.

Note: When stored at 2–6°C, the SYTOX™ AADvanced™ Dead Cell Stain working solution may be subjected to many freeze-thaw

cycles without reagent degradation.

4. Induce apoptosis in cells using the desired method. Prepare a negative control by incubating cells in the absence of apoptosis

inducing agent.

5. Harvest the cells after the incubation period and wash them in cold phosphate-buered saline (PBS).

2 Paciﬁc Blue™ Annexin V/SYTOX™ AADvanced™ Apoptosis Kit for Flow Cytometry User Guide

6. Centrifugre the washed cells, discard the supernatant, and resuspend the cells in 1X annexin-binding buer (prepared in step2) at

∼1 × 106 cells/mL, preparing a sucient volume to have 100 µL per assay.

7. Add 5µL of Paciﬁc Blue™ Annexin V (Component A) and 1µL of 500µM SYTOX™ AADvanced™ Dead Cell Stain working solution

(prepared in step3) to each 100µL of cell suspension.

8. Incubate the cells at room temperature for 30 minutes, protected from light.

9. After the incubation period, add 400 µL of 1X annexin-binding buer, mix gently, then keep the samples on ice.

10. As soon as possible, analyze the stained cells by ﬂow cytometry, measuring the ﬂuorescence emission using a 450nm bandpass

or equivalent with 405 nm excitation (Paciﬁc Blue™ dye) and a 670 bandpass or equivalent with 488 nm excitation (SYTOX™

AADvanced™).

Note: The sample can contain three populations: live cells showing a low level of violet and red ﬂuorescence, apoptotic cells

showing a high level of violet ﬂuorescence and no red ﬂuorescence, and necrotic cells showing a high intensity red and violet

ﬂuorescence (see Figure1).

11. Conﬁrm the ﬂow cytometry results by viewing the cells under a ﬂuorescence microscope using the appropriate ﬁlters.

Thermo Fisher Scientific Pacific Blue Annexin V/SYTOX AADvanced Apoptosis Kit User guide

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